WO1997018816A2 - Sulfate conjugates of ursodeoxycholic acid, and their beneficial use in inflammatory disorders and other applications - Google Patents

Abstract

One aspect of this invention is directed to a pharmacologically acceptable composition including a sulfate of 3 alpha, 7 beta-dihydroxy-5 beta-cholan-24-oic acid (Ursodeoxycholic acid or 'UDCA') and a pharmacologically acceptable carrier. In a preferred composition, the sulfate is UDCA-3-sulfate, UDCA-7-sulfate, UDCA-3,7-disulfate, glyco-UDCA-3-sulfate, glyco-UDCA-7-sulfate, glyco-UDCA-3,7-disulfate, tauro-UDCA-3-sulfate, tauro-UDCA-7-sulfate, tauro-UDCA-3,7-disulfate or a combination thereof. Another aspect of the invention concerns a method of delivering UDCA to a mammal to inhibit or treat a disorder, which includes administering a sulfate of UDCA to the mammal in an amount sufficient to inhibit or treat the disorder. For example, a UDCA sulfate may be used to advantage in inhibiting or treating an inflammatory condition of the gastrointestinal tract, such as colon cancer, rectum cancer, a neoplasm of the colon, a neoplasm of the rectum, carcinogenesis of the colon, carcinogenesis of the rectum, ulcerative colitis, an adenomatous polyp, familial polyposis and the like. A sulfate of UDCA also may be administered to inhibit or treat an inflammatory disorder of the liver. A UDCA sulfate may be used to improve serum biochemistries of liver disease or liver function, to increase bile flow or to decrease biliary secretion of phospholipid or cholesterol. In yet a further aspect, the invention is directed to a method of maintaining an isolated organ by perfusing the organ with a sulfate of UDCA.

Description

SULFATE CONJUGATES OF URSODEOXYCHOLIC ACID, AND THEIR BENEFICIAL USE IN INFLAMMATORY DISORDERS AND OTHER APPLICATIONS

Background of the Invention

3 alpha, 7 beta-dihydroxy-5 beta-cholan-24-oic acid

("Ursodeoxycholic acid" or "UDCA") has been used clinically for more

than two decades, initially proving effective for the treatment of

patients with cholelithiasis and more recently showing promise in the

treatment of patients with cholestatic liver diseases. It is well

established that oral administration of UDCA leads to a significant

improvement in serum liver enzymes, and based on results from long-

term clinical trials, the consensus opinion is that UDCA is beneficial

for the treatment of early-stage primary biliary cirrhosis. In addition,

clinical trials have shown that UDCA is beneficial in improving clinical and biochemical indices of hepatic function in patients with

sclerosing cholangitis, cystic fibrosis and chronic hepatitis.

Despite the promising effects shown by UDCA in liver

diseases, the exact mechanism of its action remains unclear. Early

speculation suggested that a shift in the hydrophobic/hydrophilic

balance of the biliary bile acid pool was an important determinant of

its effectiveness, but recent data do not totally support this

contention; and the improvement in liver function is almost certainly

the result of a marked hypercholeresis induced by UDCA, which

facilitates the biliary excretion of potentially more toxic bile acids or

other endogenous agents.

In studies focussing on the metabolism of UDCA in

patients with a variety of liver diseases, the appearance of

substantial amounts of the C-3 sulfate ester of UDCA in the urine has

been consistently observed, and this specific metabolite has proven

to be a useful marker for UDCA compliance. In addition, animal

studies have suggested that sulfation of bile acids may represent an

important metabolic pathway for preventing cholestasis and limiting

hepatocellular damage.

The cytotoxic or membrane-damaging effect of a bile

acid is related to its physicochemical properties. Hydrophobic bile

acids are markedly more membrane damaging than hydrophilic bile

acids, and relative indices of cytotoxicity have been established

based on the retention volume of the bile acid in reverse-phase high- pressure liquid chromatography systems or from partition coefficients

in octanol/water. It is paradoxical that the human liver synthesizes

chenodeoxycholic acid, a hydrophobic molecule that is intrinsically

hepatotoxic, as one of its primary bile acids; in cholestasis, the

hepatic accumulation of this bile acid may initiate, contribute to, or

exacerbate liver damage. In contrast, UDCA, the 7β-epimer of

chenodeoxycholic acid, is highly hydrophilic and has been shown to

counteract the membrane-damaging effects of hydrophobic bile acids.

This is one rationale for the therapeutic use of UDCA in the treatment

of a variety of liver diseases. After the oral or intravenous

administration of UDCA, this bile acid is efficiently biotransformed in

the liver, mainly by conjugation. Negligible concentrations and

proportions of unconjugated UDCA are consequently found in human

bile, even after the administration of relatively high doses.

UDCA also may have a therapeutic role beyond its use

in the treatment of various liver diseases. In this respect, data are

emerging from animal models of colonic carcinogenesis that suggest

a protective role for UDCA.

However, actual delivery of UDCA to the colon is

problematic, in that, at the usual therapeutic doses administered

orally ( 10-1 5 mg/kg body weight/day), UDCA is relatively well

absorbed from the intestine and efficiently biotransformed in the liver

mainly by conjugation. As a consequence, it is extremely difficult to

deliver effective amounts of UDCA specifically to the colon. Therefore, given this limitation of delivery to the colon,

it would be extremely beneficial to have a compound, composition or

method in which UDCA may be effectively delivered to the colon. It

also would be desirable to have a compound, composition or method

which may be used to deliver UDCA effectively to other portions of

the gastrointestinal tract. In addition, it would be advantageous to

have a compound, composition or method for use in effectively

inhibiting or treating an inflammatory disorder of the gastrointestinal

tract or liver.

Summary of the Invention

One aspect of this invention is directed to a

pharmacologically acceptable composition including a sulfate of 3

alpha, 7 beta-dihydroxy-5 beta-cholan-24-oic acid (Ursodeoxycholic

acid or "UDCA") and a pharmacologically acceptable carrier. In a

preferred composition, the sulfate is UDCA-3-sulfate, UDCA-7-

sulfate, UDCA-3,7-disulfate, glyco-UDCA-3-sulfate, glyco-UDCA-7-

sulfate, glyco-UDCA-3,7-disulfate, tauro-UDCA-3-sulfate, tauro-

UDCA-7-sulfate, tauro-UDCA-3,7-disulfate or a combinations thereof.

Another aspect of the invention concerns a method of

delivering UDCA to a mammal to inhibit or treat a disorder, which

includes administering a sulfate of UDCA to the mammal in an

amount sufficient to inhibit or treat the disorder. For example, a

UDCA sulfate may be used to advantage in inhibiting or treating an

inflammatory condition of the gastrointestinal tract, such as colon cancer, rectum cancer, a neoplasm of the colon, a neoplasm of the

rectum, carcinogenesis of the colon, carcinogenesis of the rectum,

ulcerative colitis, an adenomatous polyp, familial polyposis and the

like. A sulfate of UDCA also may be administered to inhibit or treat

an inflammatory disorder of the liver. A UDCA sulfate may be used

to improve serum biochemistries of liver disease or liver function, to

increase bile flow or to decrease biliary secretion of phospholipid or

cholesterol.

In yet a further aspect, the invention is directed to a

method of maintaining an isolated organ by perfusing the organ with

a sulfate of UDCA.

This invention offers several benefits and advantages

over the prior art. For example, therapeutically effective quantities of

UDCA may be delivered to the colon and other portions of the

gastrointestinal tract for inhibition or treatment of inflammatory

disorders, such as colon cancer and the like. In addition, sulfates of

UDCA may be used effectively to inhibit or treat liver disease or

improve liver function. These and other benefits and advantages will

become readily apparent to one of ordinary skill in the art upon

review of the following detailed description of the invention.

Brief Description of the Drawings

Figs. 1 A and 1 B show a comparison of the total mass of

UDCA in the entire jejunum (Fig. 1 A) and concentration in liver tissue

in control rats (Fig. 1 B) after oral administration of UDCA, UDCA-3S, UDCA-7S and UDCA-DS. Bile acids were separated according to

their mode of conjugation by anion-exchange chromatography before

analysis of the individual fractions using GC-MS. Results are

expressed as the mean values of all animals;

Fig. 2 shows fecal bile acid excretion in control rats and

rats orally administered UDCA, UDCA 3-S, UDCA 7-S and UDCA-DS.

Bile acids were separated according to their mode of conjugation by

anion-exchange chromatography before analysis of the individual

fractions using GC-MS. Results are expressed as mean values of all

animals;

Fig. 3 shows the ratio of lithocholic acid/deoxycholic

acid in the feces of control rats and rats orally administered UDCA,

UDCA 3-S, UDCA 7-S and UDCA-DS. Results are expressed as the

mean values of all animals;

Figs. 4A-4D show UDCA concentration in liver tissue of

rats orally administered UDCA (Fig. 4A), UDCA 3-S (Fig. 4B), UDCA

7-S (Fig. 4C) and UDCA-DS (Fig. 4D). Bile acids were separated

according to their mode of conjugation by anion-exchange

chromatography before analysis of the individual fractions using GC-

MS. Results are expressed as mean values of all animals;

Figs. 5A and 5B show mean biliary bile-flow and bile

acid output by Sprague-Dawley rats during IV, infusion of UDCA and

the sulfate conjugates; Figs. 6A-6D show the relationship between bile acid

secretion rate and bile-flow following infusion of UDCA and its

sulfate conjugates;

Figs. 7A and 7B show mean biliary cholesterol and

phospholipid output by Sprague-Dawley rats during IV fusion of

UDCA and the sulfate conjugates; and

Figs. 8A-8E show negative ion FAB-MS spectra of rat

bile preinfusion (Fig. 8A), during UDCA infusion (Fig. 8B), during

UDCA 3-S infusion (Fig. 8C), during UDCA 7-S infusion (Fig. 8D) and

during UDCA-DS infusion (Fig. 8E) .

Detailed Description of the Invention

Additional abbreviations appearing below include: GC

(gas-liquid chromatography), GC-MS (gas-liquid chromatography

mass spectrometry), FAB-MS (fast atom bombardment-mass

spectrometry), TLC (thin layer chromatography), HPLC (high-

performance liquid chromatography), UDCA-3S (ursodeoxycholic acid

3-sulfate), UDCA-7S (ursodeoxycholic acid 7-sulfate), UDCA-DS

(ursodeoxycholic acid 3, 7-disulfate), tBDMS ether (tert-

butyldimethylsilyl ether) and tBDMC (tert-butyldimethylsilyl chloride).

The data presented below compare the intestinal

metabolism and behavior of the individual bile acid sulfates of UDCA

with the unconjugated bile acid and show, among other things, that

the presence of the C-7 sulfate moiety protects against bacterial

degradation and inhibits intestinal absorption of UDCA. MA TERIALS AND METHODS

Synthesis of Sulfated Esters of UDCA

UDCA, 99% pure, was obtained from Sigma Chemical

Co. (St. Louis, MO). The monosulfate and disulfate esters of UDCA

were prepared by the methods described in step-by-step detail later

on in this detailed description. In brief, the synthesis of the

monosulfate esters involved selective protection of each of the ring

hydroxyl groups in the UDCA molecule, followed by sulfation of the

unprotected hydroxyl group and hydrolysis of the protecting group to

release the monosulfate ester. The disulfate conjugate of UDCA was

prepared by the reaction of UDCA with chlorosulfonic acid. Gas

chromatography - mass spectrometry (GC-MS) was used to confirm

the position of the sulfate groups by analysis of the products after

oxidation, solvolysis, and conversion to methyl/ester-trimethylsiyl

ether derivatives. Chromatographic purity of the synthetic bile salts

was found to be > 97% for UDCA 7-sulfate and UDCA 3,7-disulfate

and > 95% for UDCA 3-sulfate, as determined by high-pressure

liquid chromatography, thin-layer chromatography, and capillary-

column gas chromatography.

ANIMAL STUDIES

Male Sprague-Dawley rats (Harlan Sprague-Dawley,

Inc., Indianapolis, IN), weighing 21 0-290 g, were maintained on a

1 2-hour light-dark cycle and fed standard laboratory chow ad libitum

for 3 days. The animals were then transferred to metabolic cages in which they were housed individually and fed the same diet. UDCA,

UDCA 3-sulfate, UDCA 7-sulfate, and UDCA 3,7-disulfate were

administered by gavage at a dose of 250 mg/day for 4 consecutive

days. Each group comprised 3-6 rats. Body weights of the animals

were measured each day. On day 5, the animals killed were by

exsanguination under ether anesthesia. Plasma was collected and

frozen at -20°C. The liver was removed, rinsed in normal saline, and

flash-frozen in liquid nitrogen. Urine and feces were collected every

24 hours and frozen at -20°C. All animals received humane care in

compliance with the "Guide for the Care and Use of Laboratory

Animals" prepared by the National Academy of Sciences (National

Institutes of Health publication no. 86-23, revised in 1 985).

BILE ACID ANAL YSIS

Unconjugated and Sulfate Bile Acids in Intestinal Contents and Feces

Intestinal contents were weighed and dissected into

small pieces. In each group, feces ( 100 mg) from all animals were

pooled on day 4 of the study and then ground into a fine paste. All

samples were sonicated and sequentially refluxed in 80% methanol

for 2 hours and chloroform/methanol (1 : 1 ) for 1 hour. Samples were

taken to dryness, and the dried extracts were resuspended in 80%

methanol (20 mL) . Fractions of the methanolic extract ( 1 /40 of

intestinal contents and 1 /20 of feces samples) were removed and the

internal standard nordeoxycholic acid ( 1 0 μg) was added. This

extract was diluted with 0.01 mol/L acetic acid (20mL) and passed first through a column of Lipidex 1 000 (bed size, 4 x 1 cm; Packard

Instrument Co., Groningen, The Netherlands) and then through a

Bond-Elut C₁₈ cartridge (Analytichem, Harbor City, CA). Bile acids

were recovered by elution of the Lipidex 1 000 column and the Bond-

Elut cartridge with methanol (20 mL and 5 mL, respectively), and the

combined extracts were taken to dryness. Unconjugated bile acids

were isolated and separated from neutral sterol and conjugated bile

acids by lipophilic anion exchange chromatography on

diethylaminohydroxypropyl Sephadex LH-20 (Lipidex-DEAP; Package

Instrument Co.). Recovery of unconjugated bile acids was achieved

by elution with 0.1 mol/L acetic acid in 72% ethanol (7 mL) followed

by evaporation of the solvents. Total conjugated bile acids were

recovered with 9 mL of 0.3 mol/L acetic acid in 72% ethanol, pH

9.6. Salts were removed by passage through a Bond-Elut C_{l θ}

cartridge after addition of nordeoxycholic acid ( 1 0 μg), and

conjugated bile acids were recovered by elution with 5 mL of

methanol. Solvolysis was performed in a mixture of methanol ( 1

mL), distilled tetrahydrofuran (9 mL), and 1 mol/L trifluoracetic acid

in dioxane (0.1 mL) and heated to 45°C for 2 hours. After

solvolysis, unconjugated bile acids were isolated by chromatography

on Lipidex-DEAP. Methyl ester derivatives were prepared by

dissolving the sample in methanol (0.3 mL) and reacting with 2.7 mL

of freshly distilled ethereal diazomethane. After evaporation of the

reagents, the methyl ester derivatives were converted to trimethylsilyl ethers by the addition of 50 mL of Tri-Sil reagent

(Pierce Chemicals, Rockford, IL). A column of Lipidex 5000 (Packard

Instrument Co.) was used to remove derivatizing reagents and to

purify the sample.

Unconjugated and Sulfate Bile Acids In Plasma And Urine

In each group, urine from all animals was pooled on the

individual days of collection. Day 1 represented the baseline sample,

and samples from days 2, 3 and 4 were obtained during bile acid

administration. After the addition of nordeoxycholic acid ( 1 μg), bile

acids were quantitatively extracted from portions of urine (3 mL) and

plasma ( 1 mL) using Bond-Elut C₁₈ cartridges. After liquid-solid

extraction, isolation and separation of unconjugated and conjugated

bile acids were achieved by lipophilic anion-exchange

chromatography on Lipidex-DEAP. Solvolysis of bile acid conjugates

and isolation of the hydrolyzed products were performed as

described above, and bile acids were converted to volatile methyl

ester-trimethylsilyl ether derivatives.

Extent of Bile Acid Conjugation in Liver and Jeiunal Contents

Samples of liver ( 1 00 mg) were ground to a fine paste,

and bile acids were extracted by reflux and passage through a

column of Lipidex 1000 as described above for intestinal contents

and feces. Group separation of bile acids, according to their mode of

conjugation was achieved by lipophilic anion-exchange

chromatography. Extracts from each animal were pooled, and bile acids and their conjugates were separated using Lipidex-DEAP and

stepwise elution of the gel bed with the following buffers: 0.1 mol/L

acetic acid in 72% ethanol (unconjugated bile acids); 0.3 mol/L

acetic acid in 72% ethanol, pH 5.0 (glycine conjugates); 0.1 5 mol/L

acetic acid in 72% ethanol, pH 6.5 (taurine conjugates); and 0.3

mol/L acetic acid in 72% ethanol, pH 9.6 (sulfate conjugates) . After

evaporation of the buffers, sulfated bile acids were solvolyzed,

whereas amidated conjugates were hydrolyzed with 50 U of

cholylglycine hydrolase (Sigma Chemical Co.) in 2.5 mL of 0.2 mol/L

phosphate buffer, pH 5.6, at 37°C overnight. The resulting

unconjugated bile acids were isolated on Lipidex-DEAP, and the

methyl ester-trimethylsilyl ether derivatives were prepared as

described above.

After solvolysis, the amidated bile acids from the jejunal

portion of the rat intestine were isolated on Lipidex-DEAP by elution

with 0.1 5 mol/L acetic acid in 72% ethanol, pH 6.5 (6 mL) . Enzymic

hydrolysis was performed as described above, and the resulting

unconjugated bile acids were isolated and derivatized as described

above.

GC-MS

The methyl ester-trimethylsilyl ether derivatives were

separated on a 30 m x 0.25 mm DB-1 fused silica capillary column

(J&W Scientific, Folson, CA) using a temperature program from

225°C to 295°C with increments of 2°C/min and a final isothermal period of 30 minutes. GC-MS analysis was performed using a

Finnigan 4635 mass spectrometer (Finnigan Inc., San Jose, CA) that

housed an identical gas chromatography column operated under the

same conditions, electron ionization (70 eV) mass spectra were

recorded over the mass range of 50-800 daltons by repetitive

scanning of the eluting components. Identification of bile acids was

made on the basis of the gas chromatography retention index relative

to a homologous series of t7-alkanes, referred to as the methylene

unit value, and the mass spectrum compared with authentic

standards. Quantification of bile acids was achieved using gas

chromatography by comparing the peak height response of the

individual bile acids with the peak height response obtained from the

internal standard.

Liquid Secondary Ionization Mass Spectrometry

Liquid secondary ionization mass spectrometry negative

ion spectra of urine samples were obtained after placing

approximately 1μL of the methanolic extract onto a small drop of a

glycerol/methanoi matrix spotted on a stainless steel probe. This

probe was introduced into the ion source of a VG Autospec Q mass

spectrometer, and a beam of fast atoms of cesium, generated from

cesium iodine (35 KeV), was fired at the target containing the

sample. Negative ion spectra were obtained over the mass range of

50-1 000 daltons.

STA T/ST/CAL ANAL YSIS Data are expressed as mean ± SEM or as mean values

of all animals when extracts were pooled before analysis. Results

from different groups were compared using paired and unpaired two-

tailed Student's r-test. P values of < 0.05 were considered

statistically significant.

RESULTS

Intraluminal Bile Acid Composition Along The Intestine

The average weight of the rejected segments of intestine for the animals in the control group and those administered the individual bile acids were similar. In control animals, the total amount of UDCA in the jejunum (0.03 ± 0.01 mg) was negligible, accounting for only 0.3% of the total bile acids. However, the total mass of UDCA (and its percentage of the total bile acids) in the jejunum was greater in all animals 24 hours after the administration

of the final does of UDCA, UDCA 3-sulfate, UDCA 7-sulfate and UDCA 3,7-disulfate, accounting for 9.95 ± 0.49 mg (35.9%), 3.67 ± 0.45 mg ( 1 6.4%), 1 .09 ± 0.34 mg (4.7% and 0.21 ± 0.07 mg (2.0%), respectively. This result indicates very little conservation of UDCA when a sulfate group is conjugated in the C-7 position (Table

1 ).

Δ²² UDCA, a specific metabolite of exogenously

administered UDCA, was detected in large proportions and amounts

of the jejunum of animals administered UDCA and UDCA 3-sulfate.

However, this metabolite was not detected in the control group and accounted for < 3% of the total jejunal bile acids of the animals

administered the C-7 sulfate conjugates of UDCA (Table 1 ) .

When the conjugation pattern for UDCA in the jejunum

was examined after separation of the bile acids by lipophilic anion-

exchange chromatography (Fig. 1 ), animals administered UDCA were

found to have predominantly glyco- and tauro-UDCA, smaller

amounts of unconjugated UDCA, and negligible amounts of sulfated

UDCA. Although admidated and unconjugated forms of UDCA were

found in the jejunum after administration of the C-7 sulfates, the

total mass of UDCA in the jejunum was very small. In rats

administered UDCA-3-sulfate, the jejunum contained substantial

proportions of amidated UDCA, indicating significant

biotransformation by deconjugation and/or admidation.

With regard to endogenous bile acids, cholic acid was

the major bile acid in the jejunum of the control animals, accounting

for 48.5% ± 2.9% (6.82 ± 1 010 mg) of the total bile acids, and

after UDCA administration, there was a significant decrease {P =

0.01 ) to 1 6.8% ± 1 .3% (4.68 ± 0.65 mg). UDCA 3-sulfate also

caused a decrease (P = 0.05) in the proportion of cholic acid but to

a lesser extent than UDCA, whereas administration of the C-7

sulfates of UDCA caused an increase (P = 0.01 ) in the proportion of

cholic acid in the jejunum (Table 1 ).

In the colon of the control animals, most of the bile

acids were secondary and were identified mainly as deoxycholic and u)-muricholic acids, but only small amounts of lithocholic acid were

detected (Table 2). UDCA administration caused a decrease [P =

0.003) in the proportion of deoxycholic acid, and lithocholic acid

became the major bile acid present, accounting for 32.0% ± 0.8%

of the total colonic bile acids [P = 0.0004). In contrast, UDCA 7-

sulfate and UDCA 3,7-disulfate administration led to substantial

reductions in the mass and proportion of both deoxycholic acid and

lithocholic acid in the colon.

FECAL BILE ACID EXCRETION

No significant differences were found in the weight of

feces excreted each day among the groups of animals. Total fecal

bile acid excretion in animals administered UDCA, UDCA 3-sulfate,

UDCA 7-sulfate, and UDCA disulfate was 14.85, 1 0.70, 1 2.65 and

10.88 mg/g feces, respectively. The feces of all animals became

enriched with UDCA; however, for those animals administered the

unconjugated bile acid, UDCA was almost exclusively found in the

unconjugated form. In contrast, there were negligible concentrations

of unconjugated UDCA in the feces of rats administered UDCA 7-

sulfate and UDCA 3,7-disulfate; these two conjugates were excreted

in feces virtually unchanged (Fig. 2). The concentrations of the

major secondary bile acids excreted in feces differed among the

groups of animals, in the UDCA group, lithocholic acid increase

markedly from control values, whereas the fecal excretion of

lithocholic acid after UDCA 7-sulfate and UDCA 3,7-disulfate administration was reduced. A similar trend in deoxycholic acid

excretion was found. Compared with normal rat feces, the ration of

lithocholic acid/deoxycholic acid increased more than 20-fold when

UDCA was administered, increased 1 1 -fold with UDCA 3-sulfate

administration, and did not increase when the C-7 sulfates were

administered (Fig. 3).

Bile Acid Composition of Liver Tissue

The concentration and proportions of the individual bile

acids in liver tissue are summarized in Table 3. UDCA concentration

was 1 2.0 nmol/g in control animals, and accounted for 2.8% of the

total hepatic bile acids. Administration of UDCA and the C-3 sulfate

caused increased concentrations and proportions of liver tissue

UDCA (Fig. 1 ). In addition, Δ²² UDCA was found in the liver in

relatively high proportions. In contrast, marked decreases in the

total UDCA concentration and percent composition occurred when

animals were administered the C-7 sulfate bile acids. UDCA

administration resulted in increased hepatic lithocholic acid

concentration, whereas decreases in lithocholic acid occurred after

administration of the sulfate conjugates. Deoxycholic acid

concentration decreased in all groups with bile acid administration,

and the reduction was greater for the C-7 sulfates. Liver tissue

cholic acid concentration decreased almost 4-fold when UDCA was

administered but increased slightly after administration of the C-7

sulfate conjugates. After administration of the individual bile acids, the

conjugation of UDCA in liver tissue established that unconjugated

UDCA and its C-3 sulfate were both biotransformed by conjugation,

mainly with taurine. Irrespective of the administered bile acid,

negligible concentrations and proportions of unconjugated UDCA and

sulfated UDCA were found in the liver tissue of all animals (Fig. 4).

Bile Acid Composition of Plasma and Urine

Unconjugated plasma UDCA concentration in animals

administered UDCA was 4.8 ± 2.2 μmoi/L, and this value was

significantly greater than that found for animals administered UDCA

3-suifate (0.9 ± 0.4 mol/L), UDCA 7-sulfate (0.7 ± 0.5 μmol/L)

and UDCA disulfate (1 .0 ± 0.2 μmol/L. Sulfated UDCA

concentrations were similar and < 0.3 μmol/L in all animal groups.

The urinary excretion of UDCA was negligible ( < 0.4

nmoi/day) before bile acid administration for all animals. After

UDCA, urinary unconjugated UDCA excretion was 642.7 nmoi/day.

This was significantly greater than the concentration of UDCA

excreted in the urine of the animals administered the sulfate

conjugates (UDCA 3-sulfate, 5.2 nmoi/day; UDCA 7-sulfate, 1 .8

nmoi/day; and UDCA 3,7-disulfate, 1 .3 nmoi/day). Sulfate

conjugates of UDCA were also found in the urine after the

administration of unconjugated UDCA (4.6 nmoi/day), UDCA 3-

sulfate (2.7 nmoi/day), UDCA 7-sulfate (31 7.8 nmoi/day), and UDCA

3,7-disulfate (21 7.1 nmoi/day) . Although this represents < 0.05% of the daily dose administered, it was possible to detect these bile

acid conjugates by liquid secondary ionization mass spectrometry

analysis.

DISCUSSION

Liver tissue UDCA concentrations increased markedly

with oral administration of UDCA, and this bile acid was

predominantly conjugated by amidation. Negligible amounts of

unconjugated UDCA were found (Fig. 4), and in this regard, its

metabolism is similar to that of humans. In contrast, when the C-7

sulfate and the disulfate conjugates were administered, hepatic

concentrations of UDCA were low compared with the control

animals, indicating that these conjugates were not absorbed from the

intestine and thus, there was negligible conservation of UDCA.

Hepatic UDCA concentrations increased after administration of the

C-3 sulfate, and the fact that it was mainly amidated indicated that

significant desulfation and amidation of UDCA 3-sulfate had taken

place. The pattern of conjugation of UDCA in the jejunum paralleled

that of the liver tissue except that, in the UDCA and UDCA 3-sulfate

administered groups there was a higher proportion of unconjugated

UDCA present (Fig. 1 ). Previous studies of bile acid feeding had

established that maximum enrichment of the bile acid pool is attained

by 4 days and prolonged feeding results in no further changes in bile

acid composition. The lack of intestinal absorption of the C-7 sulfates of

UDCA is further reflected in the bile acid composition of feces. The

fecal concentration of the total amount of UDCA in the animals

administered UDCA 7-sulfate and UDCA 3,7-disulfate was markedly

greater than that found in the feces of animals administered UDCA or

UDCA 3-sulfate. In addition, the C-7 sulfates of UDCA were

predominantly excreted unchanged in feces. These findings can be

explained by the substrate specificity of bacterial sulfates, which

have been previously shown to be active only toward C-3 bile acid

sulfates. UDCA administration had a marked effect on the fecal

excretion of the major secondary bile acids and lithocholic and

deoxycholic acids. A large increase in the fecal lithocholic acid

concentration occurred when unconjugated UDCA and UDCA 3-

sulfate were administered, but UDCA 7-sulfate and UDCA 3,7-

disulfate administration had no significant effect on the fecal output

of these secondary bile acids.

The increases in fecal and hepatic lithocholic acid

concentrations can be explained by intestinal bacterial

biotransformation of UDCA and, to a lesser extent, its C-3 sulfate.

Biotransformation of UDCA to lithocholic acid occurs to a similar

extent in both rats and humans. An increase in deoxycholic acid in

the feces of animals administered UDCA is consistent with the

known competitive inhibition of cholic acid uptake at the terminal

ileum, which leads to an increased spill-over into the colon and subsequent 7α-dehydroxylation to form deoxycholic acid.

Interestingly, the UDCA C-7 sulfates seem to have the opposite

effect; cholic acid concentrations in liver tissue were increased

slightly compared with control animals, and fecal cholic acid and

deoxycholic acid concentrations were decreased.

In view of the fact that UDCA undergoes significant

biotransformation to lithocholic acid and increases fecal deoxycholic

acid concentration, both highly hydrophobic bile acids, it is perhaps

surprising that beneficial effects of UDCA have been shown in animal

models of chemically induced colon cancer. In these models, it has

been established conclusively that hydrophobic bile acids promote

tumor growth. Rectal and oral administration of bile acids, bile

diversion to the cecum, cholestyramine feeding, dietary fat, and

certain fibers, conditions that all increase the flux of bile acids

through the colon, enhance tumor formation, consistent with a

promoting effect. In vitro studies indicate that deoxycholic and

lithocholic acids are comitogenic and increase the colonic epithelial

cell proliferation rate. Other effects on ornithine decarboxylase

activity and HLA class I and II antigens have also been shown.

There are several possible explanations for the

chemopreventive effect of UDCA. Any deleterious effects of

increased lithocholic acid formation in the colon may be buffered by

the presence of relatively high concentrations of UDCA in a manner

similar to the cytopretective effects of UDCA when coincubated in vitro or coninfused in vivo with hydrophobic bile acids that are

membrane damaging. Alternatively, the protective effects may be

the result of decreased colonic deoxycholic acid concentration,

which would imply that deoxycholic acid is of major importance in

the promotion of colon cancer. Despite similar reductions in colonic

deoxycholic acid with administration of the sulfated bile acids, the C-

7 sulfates of UDCA may in principle be superior to UDCA because

these conjugates are not biotransformed to more hydrophobic bile

acids. Additionally, the lack of absorption of the C-7 sulfates in the

small intestine may permit the use of lower doses to attain similar

chemopreventive effects.

The role of bile acids in human colonic carcinogenesis is

less clear. Early studies indicate that fecal bile acid excretion,

particularly lithocholic and deoxycholic acids, was increased in

patients with colon cancer, adenomatous polyps, and familial

polyposis, although these findings were not substantiated by several

other investigators. Compared with controls, patients with colonic

cancer or adenomatous polyps have been reported to have increased

aqueous-phase lithocholic and deoxycholic acids concentrations in

feces, and these concentrations correlated with the extent of colonic

cell proliferation. Despite preliminary data supporting a

chemoprotective and/or cytoprotective effect of UDCA in animal

models of colon cancer and in vitro cell systems, the increased

lithocholic acid formation after UDCA administration may limit the overall effectiveness of UDCA in the colon. The

lithocholate/deoxycholate ration in feces is markedly increased after

UDCA and UDCA 3-sulfate administration compared with controls

(Fig. 3). This may be less desirable because the ratio of fecal

lithocholate/deoxycholate is increased in patients with colon cancer

and in patients at high risk for the disease and is proposed to be of

diagnostic value. On the other hand, UDCA 7-sulfate and UDCA 3,7-

disulfate administration resulted in no change in the

lithocholate/deoxycholate ration. Although not statistically

significant, a tendency towards a decrease in this ration was

observed, whereas the quantitative fecal excretion of these

secondary bile acids was similar to control animals.

Furthermore, as discussed above, the introduction of a

sulfate group at the position C-7 of UDCA greatly increases the

hydrophilicity of the molecule, which prevents intestinal absorption,

thereby facilitating the site-specific delivery of UDCA to the colon.

In contrast to unconjugated UDCA, which undergoes conversion to

lithocholic acid, thereby increasing the fecal lithocholic/deoxycholic

acid ratio, considered a risk factor for colonic disease, the C-7

sulfates are metabolically inert. Therefore, these conjugated bile

acids may be more effective chemoprotective agents than UDCA in

the colon. METABOLISM AND EFFECT OF SULFATE ESTERS OF RSODEOXYCHOLIC ACID ON BILE-FLOW AND BILIARY LIPID SECRETION IN RATS

The C-3 and C-7 sulfate esters and the disulfate

conjugate of UDCA were prepared as discussed in step-by-step detail

immediately below. Subsequently, the hepatic metabolism of these

bile acids in the bile fistula was examined, and these bile acids were

compared with UDCA to establish their effect on bile-flow and biliary

lipid secretion.

MATERIALS AND METHODS

Synthesis of ursodeoxycholic acid 3-sulfate (UDCA-3S)

Imidazole (3.5g) and tert-butyldimethylsilyl chloride

( 1 .6g) was added to an ice-cold solution of UDCA (2g) in anhydrous

dimethylformamide ( 1 .5ml) -pyridine (0.75ml) and the mixture was

stirred for 30 min. The reaction mixture was then poured into ice

water (20ml) and extracted with ethyl acetate (100ml) . The organic

layer was washed with water, dried over anhydrous Na₂SO₄, and

evaporated. The oily residue obtained was dissolved in hexane-ethyl

acetate (3: 1 by vol, 250 ml) and filtered through a 40g of column of

silica gel (28-200 Mesh, Aldrich Chemical Co. Inc., Wisconsin).

After evaporation of the solution the residue was dissolved in ethanol

and the product, UDCA-3-tbDMS ether (2.1 5g, yield 83%) was

crystallized. Treatment of UDCA-3tBDMS ether (2.0g) with acetic

anhydride (20mi) and pyridine (20ml) at room temperature for 5

yielded UDCA 7-acetate 3-tbDMS ether as an oily product. To a solution of UDCA 7-acetate 3-tbDMS ether in acetone (24ml) was

added 1 8% HCI (2.4ml) and the mixture was stirred at room

temperature for 30 min. The resultant product was extracted into

ethyl acetate, washed with water, dried over anhydrous Na₂SO₄, and

the solvent evaporated to give ursodeoxycholic acid 7-acetate as an

oily product. Chlorosulfonic acid ( 1 .2ml) in anhydrous pyridine

(12ml) was added to an ice cold solution of UDCA 7-acetate (1 .2g)

and the solution was heated to 50°C. After 30 minutes, the reaction

mixture was terminated by addition of water (400ml) and the product

was absorbed onto a large cartridge of octadecylsilane bonded silica,

MEGA-BOND-ELUT (Varian, Harbor City, CA) and recovered by

elution with methanol. The methanolic extract was then evaporated

to dryness and the pyridinum sale of UDCA 7-acetate 3-sulfate was

then converted to the di-sodium salt by dissolving in 0.2M

methanolic NAOH solution (40ml) and filtered. The filtrate was

diluted with cold ether (400ml), and the precipitate was collected,

washed with cold ether and dried. The solid (1 .0g) was dissolved in

MeOH (1 0ml), 3.5M NAOH (1 0ml) was added and the solution was

stirred at room temperature for 1 8h. The product was extracted by

MEGA-BOND-ELUT after diluting with water (500ml). The

methanolic extract from the cartridge was evaporated to dryness,

dissolved in 0.2M methanolic NAOH (30ml) and filtered. The filtrate

was diluted with cold ether (300ml), and the resulting precipitate

was collected and washed with ether. The procedure was repeated three times with methanol (20ml) and ether (200ml) to yield the di-

sodium salt of UDCA-3-sulfate (0,62g, yield 50%).

Synthesis of ursodeoxycholic acid 7-sulfate (UDCA-7S)

Chlorosulfonic acid (0.9 ml) in anhydrous pyridine (9 ml)

was added to an ice cold solution of UDCA 3-tbDMS ether (900 mg)

and mixture was heated to 50°C. After 30 minutes, the reaction

was terminated by addition of water (400 ml). The precipitate

pyridinum salt of UDCA 3-tbDMS 7-sulfate was washed with water,

dried under vacuum and hydrolyzed with HCI as described above.

The product was extracted with a cartridge of MEGA-BOND-ELUT

and the methanolic extract was evaporated to dryness and dissolved

in 0.2M methanolic NaOH (30 ml.). The methanolic solution was

diluted with cold ether (300 ml), and the precipitated di-sodium salt

was then isolated as described above to obtain the pure di-sodium

salt of UDCA-7-sulfate (640 mg, yield 76%).

Synthesis of ursodeoxycholic acid 3..7-disulfate (UDCA-DS)

Chlorosulfonic acid ( 1 ml) in pyridine (10ml) was added

to an ice cold solution of UDCA (1 g) in anhydrous pyridine (10ml)

and the mixture was heated to 50°C. After 60 minutes, the reaction

with terminated by addition of water (500ml). The product was

extracted with a cartridge of MEGA-BOND-ELUT and isolated as

described above to yield the di-sodium salt of UDCA-disulfate (1 .1 g,

91 % yield). Gas chromatography-mass spectrometry (GC-MS) was

used to confirm the position of the sulfate groups in all synthesized

compounds after oxidation, solvolysis and conversion to methyl

trimethylsilyl (Me-TMS) ether derivatives. Chromatographic purity of

the synthetic compounds was found to be > 97% as determined by

high-pressure liquid chromatography (HPLC), thin-layer-

chromatography (TLC) and capillary column gas chromatography

(GC).

Animal Studies

Adult male Sprague-Dawley rats (body weight 200-

230g) were anesthetized by an intraperitoneal injection of

pentobarbital (Nembutal, 7.5 mg/1 00g body weight), and maintained

under sedation by additional doses. The right jugular vein and the

common bile duct were cannulated using PE-50 polyethylene tubing

(Clay-Adams, Parsippany, N.J.) . Body temperature was maintained

throughout the experiment at 37°C using a rectal probe and a

thermostatically controlled heating pad (Harvard Apparatus Co., Inc.,

Millis, Mas.). Saline was infused at a rate of 1 .0 ml/h using a

Harvard pump (Harvard Apparatus Co., Inc.) into the jugular vein for

a control period of 2h. After collecting two 10 minute bile samples

for base-line analysis, the bile acids were individually infused

intravenously (i.v.) for 30 minutes in stepwise increasing doses (0.5,

1 .0, and 2.0 μmol /min/1 OOg body weight) . Bile acid solutions were

prepared in 3% human albumin in 0.45% saline. Six animals were used for each experiment and bile was collected every 10 minutes

into preweighed tubes. At the end of the experiment, blood was

obtained by cardiac puncture, and urine was obtained by aspiration

of the bladder. All biological specimens were stored at -20°C. This

animal study protocol (#1 B1 0044) was approved by the Bioethics

committee of the Children's Hospital Medical Center (Cincinnati,

Ohio).

Analytical Techniques

TLC was performed on precoated silica gel G plates

(Merck, 0.2 mm thickness) using a solvent system of n-butanol-

acetic acid-water ( 1 0: 1 : 1 , by vol) . The spots were visualized by

spraying with a 1 0% ethanolic solution of phosphomolybdic acid

followed by heating at 120°C for 5 minutes.

HPLC was performed using a Varian 5000 HPLC

instrument (Varian Associates Inc., Palo Alto, CA) equipped with a

variable wavelength UV detector and housing a 25 x 0.46 cm

Hypersil ODS column (5 μm particle size; Keystone Scientific,

Bellefonte, PA). The column was operated at ambient temperature

and the eluting solvent was methanol -0.01 M phosphate buffer

(65:35, by vol), adjusted to pH 6.8 and modified from the method of

Rossi et al. (High pressure liquid chromatographic analysis of

conjugated bile acids in human bile: simultaneous resolution of

sulfated and unsulfated lithocholyl amidates and the common

conjugated bile ducts. J. Lipid Res. 28: 589-595 ( 1 987)). Flow rate was 1 .0 ml/min and bile acids were detected by absorption at

205 nm.

GC was carried out on a Hewlett-Packard 5890 gas

chromatograph housing a 30 meter DB-1 (4 mm i.d.; 0.25 μm film)

fused silica capillary column (J and W Scientific inc., Rancho

Cordova, CA) and using a temperature program from 225°C to 295°C

in increments of 2°C/min with initial and final isothermal periods of 2

minute and 30 minutes respectively. Helium was used as the carrier

gas with a flow-rate of 1 .8 ml/min.

GC-MS was carried out on either a VG Autospec Q

magnetic sector instrument or a Finnigan 4635 quadruple GC-MS-DS

instrument housing identical GC columns and operated under the

same chromatographic conditions. Electron ionization (70 eV) mass

spectra were recorded over the mass range 50 to 1000 Da/e by

repetitive scanning of the eluting components.

Negative ion fast atom bombardment-mass

spectrometry (FAB-MS) spectra of bile samples, urine and synthetic

compounds were obtained after placing the equivalent of

approximately 1 μ1 of the original bile extract, 1 0 μ1 - 50μ1 of the

urine extract and μg quantities of synthetic bile acids dissolved in

methanol onto a small drop of a glycerol matrix spotted on a copper

target of the FAB probe. This probe was introduced directly into the

ion source of the mass spectrometer and a beam of fast atoms of

either xenon generated with a saddle field atom gun (Ion Tech, Teddington, Middlesex, UK) operated at 8kV and 20 μA, or cesium

generated from cesium iodine (35kV), was fired at the target

containing the sample. Negative ion spectra were obtained over the

mass range of 50-1 000 Da/e.

Bile Analysis

Bile volume was determined gravimetrically assuming a

density of 1 g/ml. Total 3α-hydroxy bile acid concentration in the

bile was measured enzymaticaliy before solvolysis (nonsulfated bile

acids) and after solvolysis (total bile acids) (Mashige, F., et al. , Direct

spectrometry of total bile acids in serum. C/t>7. Chem. 27: 1 352-

1 356 ( 1 981 )). The bile acid output was calculated by multiplying

the rate of bile-flow by the bile acid concentration. Biliary

phospholipids were determined by an enzymatic procedure based on

the choline oxidase method (Nippon Shoji Kaisha, Ltd., Osaka,

Japan) (Grantz, D., et al. , Enzymatic measurement of choline-

containing phospholipids in bile. J. Lipid Res. 22: 273-276

(1 981 )). Cholesterol was also measured enzymaticaliy (Boehringer

Mannehim, Indianapolis, IN) (Fromm, H., et al. , Use of a simple

enzymatic assay for cholesterol analysis in human bile. J. Lipid Res.

21 : 259-261 ( 1 980)).

Biliary and Urinary Bile Acid Analysis

For the determination of hepatic biotransformation of

the infused bile acid, bile collections from the six animal were pooled

and biliary bile acids were determined by GC-MS after extraction, solvolysis, hydrolysis, and derivatization. Quantification of bile acids

was achieved using GC, by comparing the peak height response of

the individual bile acid with the peak height response obtained from

the internal standard, nordeoxycholic acid added to the initial sample

of bile. Identification of a bile acid was made on the basis of the

retention index relative to a homologous series of n-alkanes, referred

to as the methylene unit value (MU) and the fragmentation pattern of

the mass spectrum was compared with authentic standards. A list

of over 100 mass spectra of authentic bile acid standards and

retention indices was recently compiled as a reference source

(Lawson, A.M., et al. , Mass spectrometry of bile acids, The Bile

Acids, Vol. 4, Methods and Applications, pp. 1 67-267 (1 988)).

Urine collections from the six animals were pooled and the bile acids

were extracted by liquid-solid extraction using a Bond Elut-C₁₈

cartridge and bile acids were analyzed by GC-MS after solvolysis,

hydrolysis and derivatization.

Group Separation of Bile Acids Using Lipophilic Anion

Exchange Chromgtography

In the case of the animals infused with UDCA-3S, bile

was collected during the final period of bile acid infusion ( 1 .0

μmol/min/100g body weight). Bile acids were solvolyzed and

separated into groups based on their mode of conjugation using the

lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex

LH-20; Packard Instruments, Groningen, The Netherlands). Bile acid composition was determined in each fraction by GC-MS after

hydrolysis and preparation of the Me-TMS ethers.

Statistical Methods

Results were expressed as mean ± standard error of

mean (SEM). Bile-flow and biliary lipid output were expressed as

μ1 /min/g liver and nomol/min/g liver, respectively. Statistical

analysis was made using INSTAT program (Graphpad Software Inc.,

San Diego, CA) . Parametric data among groups were analyzed using

Student's t-test. The statistical comparisons between the different

groups were made by one-way analysis of variance (ANOVA) . When

the values were found to be significant with respect to infusions of

different bile salts, the comparison of any of two groups were made

by Bonferroni's t-test. Linear regression analysis was performed.

The choleretic activity of each bile acid was determined from the

slope of regression line of the correlation between the bile acid

secretion rate and bile-flow and was expressed as μl /μmol.

Comparisons between slopes were made by one-way ANOVA.

RESULTS

Bile-Flow and Bile Acid Secretion

The effects of i.v. infusion of UDCA, UDCA-3S, UDCA-

7S and UDCA-DS on bile-flow and bile acid secretion rate are

depicted in Figs. 5A and 5B and summarized in Table 4. All bile

acids were markedly choleretic and the order of maximum bile-flow

was UDCA-3S < UDCA < UDCA-7S = UDCA-DS. Biliary bile acid secretion rate increased in all animals during infusion of UDCA-3S,

UDCA, UDCA-DS and UDCA-7S to a maximum of 1 38.9 ± 1 1 .8,

1 45.1 ± 1 7.3, 222.4 ± 24.3, and 255.4 ± 1 8.2 nmol/min/g liver,

respectively. For comparison, basal bile acid secretion averaged

40.4 ± 4.2 nmol/min/g liver. The relationship between bile acid

secretion rate and bile-flow following infusion of each bile acid is

shown in Fig. 6. The apparent choleretic activities of UDCA, UDCA-

7S, UDCA-3S and UDCA-DS calculated from the slopes of the

regression lines were, 1 6 ± 2, 1 5 ± 1 , 1 3 ± 1 and 1 6 ± 1 μ1 /μmol,

respectively. The intercepts of the lines indicated bile acid-

independent bile-flow was of the same magnitude ( 1 .7μ1 /min/g liver)

for all the groups of animals.

Biliary Lipid Secretion

The effects of infusing UDCA and its sulfate conjugates

on the biliary output of cholesterol and phospholipids are shown in

Figs. 7A and 7B. Over the first 40 minutes of infusing UDCA and

UDCA-7S, biliary cholesterol increased, attaining a maximum

secretion rate of 1 .88 ± 0.1 9 and 1 .76 ± 0.21 nmol/min/g liver

respectively and cholesterol secretion was maintained with UDCA-7S

but showed a significant decline when UDCA was infused even with

a stepwise increase in dose. In the pre-infusion periods, the

corresponding secretion rates for cholesterol averaged 1 .46 ± 0.1 and

1 .42 ± 0.1 6 nmol/min/g liver respectively. By contrast, infusions of

UDCA-3S and UDCA-DS significantly reduced the biliary cholesterol output to 0.40 ± 0.05 and 0.37 ± 0.1 2 nmol/min/g liver respectively

compared with the basal values. Biliary phospholipid secretion

increased significantly with UDCA and UDCA-7S infusion, but by

contrast, UDCA-3S and UDCA-DS caused a significant reduction in

biliary phospholipids (Table 4).

Negative Ion FAB-MS Analysis Of Biliary And Urinary Bile Acids

Negative ion FAB-MS spectra of the pooled rat bile

collected before (basal period) and during infusion of unconjugated

UDCA are shown in Figs. 8A and 8B. The ions of m/z 51 4 and m/z

498 in the basal bile samples represent the taurine conjugates of

trihydroxy- and dihydroxy-cholanoates respectively and represent

primary bile acids which are major species in rat bile. During infusion

of UDCA, the predominant ions in the spectrum became m/z 498 and

m/z 448 indicating that infused UDCA was almost exclusively

conjugated with taurine and glycine. The ion at m/z 471 indicates a

dihydroxycholanoate (UDCA) sulfate while those at m/z 567 and m/z

589 (sodium adduct) represent flucuronide conjugates of UDCA.

During infusion of UDCA-7S, the predominant ions in

the spectrum (Fig. 8D) were m/z 471 and its sodium adduct m/z

493, and these ions represent unchanged UDCA-7S. No other

significant ions were present to indicate further metabolic

transformation of UDCA-7S.

During infusion of UDCA-3S, ions at m/z 471 and m/z

493 (sodium adduct) confirmed biliary secretion of the unchanged bile acid and the ions at m/z 550 and m/z 600 reflect amidation with

glycine and taurine (Fig. 8C).

During infusion of UDCA-DS, the predominant ions were

m/z 471 and m/z 493 (sodium adduct) and m/z 573. The ion of m/z

573 represents UDCA-DS, and m/a 471 and m/z 493 are fragment

ions. No other ions were present to suggest further metabolic

transformation of UDCA-DS (Fig. 8E).

GC-MS Analysis of Biliary Bile Acids

Table 5 summarizes the relative percentage composition

of individual bile acids in bile following infusion of UDCA and the

various sulfate conjugates. In the basal state, cholic acid, α-

muricholic and β-muricholic acids were major bile acids of rat bile.

During infusion of all of the compounds, UDCA became the

predominant biliary bile acid and the percentage composition was

similar among all groups, indicating that all of the sulfated bile acids

were taken up by the liver and efficiently secreted into bile.

There was no evidence to support any biotransformation

including amidation of the C-7 sulfate and the disulfate esters of

UDCA, however both unconjugated UDCA and UDCA-3S were

metabolized by further hydroxylation, most probably in the side-

chain, however the exact structure of this hydroxlated metabolite

remains to be definitively established.

Separation of bile acid conjugates secreted in bile during

infusion of UDCA-3S indicated that equal proportions (25%) of the infused bile acid were recovered as taurine and glycine conjugates,

while UDCA was almost exclusively conjugated with taurine and

glycine. Because of the lack of biotransformation of UDCA-7S and

UDCA-DS, confirmed by FAB-MS, further conjugate separation

studies were deemed to be unnecessary.

Urinary Bile Acid Analysis

Negative ion FAB-MS analysis of all urine samples

indicate that the sulfate esters of UDCA were all excreted in urine,

and this was confirmed by GC-MS analysis. Quantitatively,

however, the relative proportion of UDCA sulfate excreted in urine

was small compared with the biliary excretion which was the major

route of elimination. When UDCA was infused negligible proportions

(0.01 %) of the total dose administered appeared in the urine. For

UDCA-3-sulfate, UDCA-7-sulfate and UDCA-disulfate, the

corresponding proportions of the administered doses appearing in

urine were 2.8%, 0.9% and 2.2% respectively.

DISCUSSION

The results presented above demonstrate that, like

UDCA, all of the sulfate conjugates are markedly choleretic and

increase bile acid secretion. The order of maximum bile-flow for the

individual bile acids was UDCA-DS = UDCA-7S > UDCA > UDCA-

3S, and was not directly related to their relative

hydrophobic/hydrophilic nature as determined from the HPLC

retention indices. The presence of a sulfate moiety in the C-7 position of

the bile acid nucleus results in a significantly higher bile-flow than

that induced by UDCA, while the C-3 sulfate, although being

choleretic, was less effective in stimulating bile-flow than

unconjugated UDCA. These differences might be explained by the

fact that significant amidation of the C-3 sulfate takes place during

first-pass hepatic clearance, whereas a sulfate moiety at the position

C-7 prevents biotransformation. It is possible that the amidated

sulfates have lower choleretic properties. Interestingly the relative

proportions of UDCA appearing in the bile were similar for all of the

bile acids examined even though there were significant differences in

bile-flow among these compounds.

With regard to cholesterol and phospholipid secretion a

clear trend was evident. The bile acids that were the most polar

(evidenced by their HPLC retention indices) were found to cause a

significant decrease in biliary cholesterol and phospholipid output.

This relationship between bile acid hydrophobicity/hydrophilicity and

cholesterol and phospholipid secretion is most probably associated

with the detergenicity of the molecule, i.e., the less detergent and

highly polar bile acid sulfates are less membrane damaging than the

more hydrophobic bile acids.

The combined effects of a lower cholesterol and

phospholipid secretion and greater hypercholeresis induced by the

highly hydrophilic 3-sulfate and 3, 7-disulfate conjugates of UDCA compared with unconjugated UDCA would suggest that these

particular bile acid sulfates might be more efficacious agents for the

treatment of cholestatic liver disease.

Marked differences in hepatic biotransformation of the

individual UDCA sulfates were observed. For example, substitution

of a sulfate moiety at position C-7 in the nucleus hindered hepatic

biotransformation so that UDCA-7-sulfate and UDCA-disulfate were

both secreted into bile unchanged. This was not the case for the C-3

sulfate of UDCA, which was secreted into bile to a limited extent

unchanged, but also underwent appreciable amidation with taurine

and glycine and further hydroxylation, most probably in the side-

chain. Unconjugated UDCA on the other hand was mainly

conjugated with taurine, and to a lesser extent was converted to

glycine, sulfate and flucuronide conjugates before biliary secretion.

Negligible amounts of UDCA sulfates were excreted in

the urine even following infusion of relatively high concentrations.

This was particularly surprising when one considers that the majority

of urinary bile acids are sulfate conjugates. The lack of biliary bile

acid sulfates in patients with cholestatic liver disease and the finding

of high proportions and high concentrations of sulfated urinary bile

acids can therefore only be explained by renal sulfation, and not

hepatic sulfation of bile acids. These observations clearly

demonstrate that sulfated bile acids are readily taken up by the liver

and transported into bile, and therefore, would not appear to support the generally held belief that hepatic sulfation is an important

metabolic pathway in cholestasis. In this respect, the kidney may be

an important metabolic organ in protecting the liver from the toxicity

of bile acids during cholestasis.

The rat is a species that significantly 6β-hydroxylates

bile acids, and 6β-hydroxylation of CDCA and UDCA has been

shown to occur. In this study, significant amounts of hydroxylated

products of UDCA, such as muricholic acid isomers, could not be

detected. It has been reported that taurochenodeoxycholic acid

disulfates and glycochenodeoxycholic acid disulfates were

metabolized by 90% to 3α, 7α-disulfate, 6β-hydroxy 5β-cholanoic

acid (3α, 7α-disulfate of α-muricholic acid) in bile fistula rats. In the

experiments conducted with regard to the invention, neither UDCA-

7S nor UDCA-disulfate were hydroxylated. These experiments

strongly suggest that the presence of the sulfate group prevents

hepatic biotransformation of the nucleus. However, it might be

possible that the enzyme responsible for hydroxylation preferentially

acts upon the amidated bile acids as substrates.

It is thought that conjugation of bile acids with taurine

depends on the substrate affinity of the enzyme (bile acid

CoA:glycine/taurine-N-acyl-transferase) and the supply of taurine in

the liver. Our results of FAB-MS and GC-MS showing that UDCA-3S

was amidated partially with taurine and glycine, indicates the sulfate

ester has less affinity for the enzyme compared with the nonsulfate bile acid. The observation that UDCA-7S and UDCA-DS were not

amidated with taurine or glycine indicates that the presence of a 7β-

sulfate moiety hinders the enzyme activity.

NOTE RESULTS ARE EXPRESSED AS MEAN ± SEM ND NOT DETECTED

-

NOTE: RESULTS ARE EXPRESSED AS MEAN ± SEM ND: NOT DETECTED

NOTE RESULTS ARE EXPRESSED AS MEAN ± SEM ND NOT DETECTED

TABLE 4. Physiological effects of infusion of UDCA and its sulfate conjugates

Cholesterol outputt (nmol/min/g liver)

Basal 1.46 1 0.10 1.42 ± 0.16 1.28 t 0.05 1.52 ± 0.12

Maximal change 1.88 ± 0.19 1.76 i 0.23 0.40 ± 0.02 0.37 ± 0.07

Phospholipid outputt (nmol/min/g liver)

Basal 10.30 t 0.70 9.00 ± 1.00 9.60 ± 0.80 9.80 ± 0.70

Maximal change 17.20 ± 1.90 15.00 i 1.70 4.00 ± 0.50 2.50 ± 0.30

Values are expressed as mean ± SEH for six animals in each group.

Although several particular aspects of the invention

have been discussed in detail above, the scope of the invention is

not limited to these aspects, and instead, is to be determined by the

following claims.

Claims

WHAT IS CLAIMED IS:

1 . A pharmacologically acceptable composition,

comprising:

a sulfate of 3 alpha, 7 beta-dihydroxy-5 beta-cholan-24-

oic acid (UDCA); and

a pharmacologically acceptable carrier.

2. The pharmacologically acceptable composition of claim

1 wherein said sulfate is selected from the group consisting of

UDCA-3-sulfate, UDCA-7-sulfate, UDCA-3,7-disulfate, glyco-UDCA-

3-sulfate, glyco-UDCA-7-sulfate, glyco-UDCA-3,7-disulfate, tauro-

UDCA-3-sulfate, tauro-UDCA-7-sulfate, tauro-UDCA-3,7-disulfate

and combinations thereof.

3. The pharmacologically acceptable composition of claim

1 wherein said sulfate is selected from the group consisting of

UDCA-3-sulfate, UDCA-7-sulfate, UDCA-3,7-disulfate and

combinations thereof.

4. The pharmacologically acceptable composition of claim

1 wherein said sulfate is selected from the group consisting of

UDCA-7-sulfate, UDCA-3,7-disulfate and combinations thereof.

5. The pharmacologically acceptable composition of claim

1 wherein said sulfate is present in an amount effective to inhibit or

treat an inflammatory condition of the gastrointestinal tract.

6. The pharmacologically acceptable composition of claim

5 wherein said inflammatory condition is selected from the group

consisting of an inflammatory condition of the small intestine, an

inflammatory condition of the large intestine and combinations

thereof.

7. The pharmacologically acceptable composition of claim

5 wherein said inflammatory condition is selected from the group

consisting of colon cancer, rectum cancer, a neoplasm of the colon,

a neoplasm of the rectum, carcinogenesis of the colon,

carcinogenesis of the rectum, ulcerative colitis, an adenomatous

polyp, familial polyposis and combinations thereof.

8. The pharmacologically acceptable composition of claim

1 wherein said sulfate is present in an amount effective to inhibit or

treat an inflammatory condition of the liver.

9. The pharmacologically acceptable composition of claim

1 wherein said sulfate is present in an amount effective to deliver

UDCA to the large intestine of a mammal.

10. The pharmacologically acceptable composition of claim

9 wherein said sulfate includes a sulfate moiety on the C-7 carbon,

said sulfate present in an amount effective to improve delivery of

UDCA to the large intestine of a mammal, relative to delivery of

UDCA to the large intestine by UDCA or a sulfate of UDCA without a

sulfate moiety on the C-7 carbon.

1 1 . The pharmacologically acceptable composition of claim

1 for the inhibition of the intestinal absorption of UDCA.

1 2. The pharmacologically acceptable composition of claim

1 for the inhibition of the intestinal transformation of UDCA and its

metabolites.

1 3. The pharmacologically acceptable composition of claim

1 2 for the inhibition of the intestinal transformation of UDCA and its

metabolites by bacterial degradation.

14. The pharmacologically acceptable composition of claim

1 3 for the inhibition of the intestinal transformation of UDCA and its

metabolites by 7 alpha-dehydroxylation.

1 5. The pharmacologically acceptable composition of claim

1 wherein said sulfate includes a sulfate moiety on the C-7 carbon,

said composition for decreasing the amount of lithocholic acid or a

salt thereof and deoxycholic acid or a salt thereof in the colon.

1 6. The pharmacologically acceptable composition of claim

1 wherein said sulfate includes a sulfate moiety on the C-7 carbon,

said composition for the delivery of UDCA to the colon without

substantially increasing the ratio of lithocholic acid or a salt thereof

to deoxycholic acid or a salt thereof in the colon.

1 7. The pharmacologically acceptable composition of claim

1 for the improvement in serum biochemistries of liver disease and

liver function.

1 8. The pharmacologically acceptable composition of claim

1 7 wherein said serum biochemistries include serum concentrations

of an enzyme selected from the group consisting of alanine

aminotransferase, aspartate aminotransferase, alkaline phosphatase,

gamma-glutamyltranspeptidase and combinations thereof.

1 9. The pharmacologically acceptable composition of claim

1 for increasing bile flow.

20. The pharmacologically acceptable composition of claim

1 for decreasing biliary secretion of a lipid selected from the group

consisting of phospholipid, cholesterol and combinations thereof.

21 . The pharmacologically acceptable composition of claim

1 wherein said composition is formulated for perfusion of an isolated

organ.

22. The pharmacologically acceptable composition of claim

21 wherein said isolated organ is selected from the group consisting

of a liver, a lung, a kidney and an intestine.

23. The pharmacologically acceptable composition of claim

1 formulated for oral, local or intravenous administration.

24. The pharmacologically acceptable composition of claim

8 formulated for intravenous administration.

25. A method of delivering 3 alpha, 7 beta-dihydroxy-5

beta-cholan-24-oic acid (UDCA) to a mammal to inhibit or treat a

disorder, comprising the step of:

administering a sulfate of UDCA to said mammal in an

amount sufficient to inhibit or treat said disorder.

26. The method of claim 25 wherein said sulfate is selected

from the group consisting of UDCA 3-sulfate, UDCA-7-sulfate,

UDCA-3,7-disulfate, glyco-UDCA-3-sulfate, glyco-UDCA-7-sulfate,

glyco-UDCA-3,7-disulfate, tauro-UDCA-3-sulfate, tauro-UDCA-7-

sulfate, tauro-UDCA-3,7-disulfate and combinations thereof.

27. The method of claim 25 wherein said sulfate is selected

from the group consisting of UDCA-3-sulfate, UDCA-7-sulfate,

UDCA-3,7-disulfate and combinations thereof.

28. The method of claim 25 wherein said sulfate is selected

from the group consisting of UDCA-7-sulfate, UDCA-3,7-disulfate

and combinations thereof.

29. The method of claim 25 wherein said disorder is an

inflammatory condition of the gastrointestinal tract.

30. The method of claim 29 wherein said inflammatory

condition is selected from the group consisting of an inflammatory

condition of the small intestine, an inflammatory condition of the

large intestine and combinations thereof.

31 . The method of claim 29 wherein said inflammatory

condition is selected from the group consisting of colon cancer,

rectum cancer, a neoplasm of the colon, a neoplasm of the rectum,

carcinogenesis of the colon, carcinogenesis of the rectum, ulcerative

colitis, an adenomatous polyp, familial polyposis and combinations

thereof.

32. The method of claim 25 wherein said disorder is an

inflammatory condition of the liver.

33. The method of claim 25 wherein said sulfate is

administered in an amount sufficient to deliver UDCA to the large

intestine of said mammal.

34. The method of claim 33 wherein said sulfate includes a

sulfate moiety on the C-7 carbon, said sulfate administered in an

amount effective to improve delivery of UDCA to the large intestine

of said mammal, relative to delivery of UDCA to the large intestine by

UDCA or a sulfate of UDCA without a sulfate moiety on the C-7

carbon.

35. The method of claim 25 wherein said method inhibits

the intestinal absorbtion of UDCA.

36. The method of claim 25 wherein said method inhibits

the intestinal transformation of UDCA and its metabolites.

37. The method of claim 36 wherein said method inhibits

the intestinal transformation of UDCA and its metabolites by bacterial

degradation.

38. The method of claim 37 wherein said method inhibits

the intestinal transformation of UDCA and its metabolites by 7 alpha-

dehydroxylation.

39. The method of claim 25 wherein said sulfate includes a

sulfate moiety on the C-7 carbon, said sulfate administered in

amount sufficient to decrease the amount of lithocholic acid or a salt

thereof and deoxycholic acid or a salt thereof in the colon.

40. The method of claim 25 wherein said sulfate includes a

sulfate moiety on the C-7 carbon, said sulfate administered in an

amount sufficient to deliver UDCA to the colon without substantially

increasing the ratio of lithocholic acid or a salt thereof to deoxycholic

acid or a salt thereof in the colon.

41 . The method of claim 25 wherein said disorder is a

disorder of the liver, said sulfate administered in an amount sufficient

to improve serum biochemistries of liver disease and liver function.

42. The method of claim 41 wherein said serum

biochemistries include serum concentrations of an enzyme selected

from the group consisting of alanine aminotransferase, aspartate

aminotransferase, alkaline phosphatase, gamma-

glutamyltranspeptidase and combinations thereof.

43. The method of claim 25 wherein said sulfate is

administered in an amount sufficient to increase bile flow.

44. The method of claim 25 wherein said sulfate is

administered in an amount sufficient to decrease biliary secretion of a

lipid selected from the group consisting of phospholipid, cholesterol

and combinations thereof.

45. The method of claim 25 wherein said sulfate is

administered by a method selected from the group consisting of oral

administration, intravenous administration and combinations thereof.

46. The method of claim 32 wherein said sulfate is

administered by intravenous administration.

47. A method of maintaining an isolated organ, comprising

the step of:

perfusing said isolated organ with a sulfate of 3 alpha, 7

beta-dihydroxy-5 beta-cholan-24-oic acid (UDCA) .

48. The method of claim 47 wherein said isolated organ is

selected from the group consisting of a liver, a lung, a kidney and an

intestine.